A UV-vis spectrophotometer comprises a light source, a monochromator, a sample cuvette (cell), and a photodetector. There are two types of optical arrangement: single beam and double beam (Fig. 4). In either case, white light from a light source (LS) is introduced into a monochromator and dispersed by a diffraction grating. Only light with a specific wavelength is extracted from the resulting spectrum through the exit slit. In the case of a single beam, the obtained monochromatic light with intensity I0 irradiates a sample and the transmitted light with intensity I is detected. Here, I/I0 is the transmittance. In the double-beam configuration, the monochromatic light is divided into two by a beam splitter, and the individual beams pass through a sample and a reference, following which both are detected. By bifurcating the optical path, both I0 and I can be measured simultaneously.
Fig. 4 UV-vis spectrophotometer configuration (upper: single beam, lower: double beam)
Beer’s law forms the basis of quantification using absorption spectroscopy. Here, the intensity of incident light in a sample cuvette is I0 and the intensity of transmitted light is I, and
I = I0 e –εC L
where C is the molar concentration of the solution, L is the thickness, and ε is the molar extinction coefficient
Taking the logarithm on both sides and transforming the formula,
-log (I / I0 ) = εCL
If the left side -log(I / I0) is defined as the absorbance A, then
A = εCL
That is, the absorbance A is proportional to the concentration C of the sample (Fig. 5). This equation is a calibration curve, and it can be used to accurately determine the concentration of unknown substances.
Fig. 5 Calibration curve for quantitative analysis
The photometric modes (the vertical axis of the spectrum) used in UV-vis spectroscopy are:
Absorbance: Abs = log (I0 / I)
Transmittance: %T = I / I0 × 100
Reflectance: %R = I / I0 × 100
The experimental setup for transmission and reflection measurements is shown in Fig. 6.
Fig. 6 Transmission measurement (%T) and reflection measurement (%R)
When measuring a sample, Abs is used for quantitative analysis of factors such as concentration and turbidity, and %T and %R are used to determine factors such as the transmission/reflection characteristics, film thickness, and color.
A deuterium lamp (185 to 400 nm) is used for the ultraviolet region and a halogen lamp (350 to 3000 nm) is used for the visible / near infrared region. In some cases, a xenon flash lamp is also used. This kind of lamp is often employed together with a photodiode array (PDA) detector in HPLC.
The monochromator extracts light with a specific wavelength from a spectrum produced by a dispersion element such as a diffraction grating. The monochromator comprises an entrance slit, a collimator mirror, a diffraction grating, a condenser mirror, and an exit slit. Figure 10 shows the optical configuration of a typical Czerny-Turner type monochromator.
Fig. 10 Czerny-Turner monochromator
There are many types of commercially available cuvettes. These can be made from glass, quartz, or plastic, and can have optical path lengths from 5 to 100 mm. In addition to cuvettes with rectangular cross sections, shapes such as capillaries and flow cells are also available.
The detector in a UV/vis spectrophotometer is a transducer that converts light into an electrical signal. Typical examples include Si photodiodes (190 – 1100 nm) and photomultiplier tubes (PMT, 185 – 900 nm). PbS detectors (1000 – 3200 nm) and InGaAs detectors (1000 – 3200 nm) are used as near-infrared detectors.